1. Ukraine Clinical Project Progress
April 7-8, 2014

2. Aims 1 & 2
Recall mothers and children for visits at 2 yrs and yrs of age
Interviews
Growth
Food records
Biological sampling
3D imaging
Cardiac orienting response paradigm
Preschool testing battery at yr visit

3. Aims 1 & 2
Recruit an additional 120 pregnant women with same follow-up as Phase II
Incorporate these children/mothers into the Phase III protocol

4. Aims 1 & 2: Phase III Visits
Visit Under Phase III Protocol
Number of Visits
Number of Unique Subjects
New pregnant women enrolled Phase III
137
Mothers re-consented in Phase III for new data collection 42
Dysmorphology exams completed
325
223
Prenatal ultrasounds completed
240
139
Cardiac orienting paradigm completed
205
113
3D images captured 72
2 year old visits completed 17
3.5-5 year old visits completed 25

5. On-Site Training Ukrainian Staff on Administration and Scoring of Preschool Test Protocol.
Developed Preschool Battery for Assessment of Cognitive and Behavioral Effects of Prenatal Alcohol Exposure
Sensitive to Effects of Prenatal Exposure and Consistent with CIFASD Neurobehavioral Battery
Suitable for Children 3 ½ to 5 years
Not reliant on Verbal Skills to avoid Language Confounds
Acceptable to Ukrainian Staff and Research Participants
Perceived by Ukrainian colleagues to have clinical merit
Assuring Reliability and Validity in Test Administration and Scoring.
Repeated On Site Training
Review of records of Test Administrations
Regular Skype “conferences”

6. Brain Involvement in Orienting Responses
The heart receives input from the 10th cranial nerve via the medulla oblongata
Electrical stimulation in animal (rabbit) models of the vmPFC (ventral-medial prefrontral cortex) and DLPFC (dorsal lateral prefrontal cortex) result in ORs
Mediodorsal nuclei of the thalamus and the intralaminar nucleus of the thalamus are also involved in OR responses (Powel, Watson, & Buchanan, 1990)
Cardiac orienting responses index early frontal lobe functioning

7. Improvements in the New Neurophysiological Protocol
Expanded to 2 sites
Upgraded equipment to Biopac’s Wireless Bioharness and trained staff to carry out the protocol

8. Preschool Physiology Protocol
Repeating Auditory OR Response (5/3)
Three Little Pigs (Auditory/Visual Habituation Combined 3/3)
Auditory stimuli:
Habituation: hertz pure tone pairs presented contiguously for 12 sec each
Dishabituation: hertz pure tone pairs presented contiguously for 12 sec each

9. Reaction Time Task
Looks at sustained attention while performing a reaction time task.
During sustained attention HR and HR variability is reduced
Children press a special preschool mouse when they see a picture

10. Discriminate Function Analysis-Combining All ORs
Auditory
Future Goals
Determine predictive validity to 12-months Bayleys
Determine predictive validity to preschool period
Improve mathematical modeling of the dynamic OR response and determine if this improves prediction
Discriminate Function Analysis-Combining All ORs
6-Month Classification
Predicted
Normal
Delay 91 9 2 10
90.2 % Classified Correctly
Specificity (% of persons without delay who test negative)
Sensitivity (% of persons with delay who test positive)
Habituation
Dishabituation

11. Aim 3: miRNA
142 archived maternal plasma samples selected and shipped to Texas A&M
71 mothers in 3 groups sampled:
two available maternal blood samples per subject
at least one physical exam of the child
at least one completed neurobehavioral test
samples are classified into three groups
exposed mothers who had children with at least one physical feature of FASD and at least one Bayley score <85
exposed mothers who had children with no physical features of FASD and no Bayley score <85
unexposed/low exposed mothers

12. Aim 3: miRNA
Initial quality control assessment of samples has been performed for 60 plasma samples and deemed adequate. Dr. Miranda’s lab has isolated RNA from a total of 24 samples and has been able on average to recover ~1000 ng RNA per sample
There are no statistically significant differences in RNA recovery due to recruitment site, stage of pregnancy or treatment group

13. A B p<0.04 Plasma Erythrocytes Human Plasma Samples Plasma Peaks
TCR1944 3rd Trimester
TCR1944 1st Trimester
Sheep
Free
Hemoglobin
Deoxy- and oxy-hemoglobin
Plasma
Erythrocytes

14. Aim 4: Genetics/Epigenetics
500 WBC maternal samples banked at UCSD for assessment of methylation status
Selecting samples based on criteria for miRNA and likelihood that paired child sample will be available
Plan to process all selected samples in one batch

15. Aim 4: 3D images (one site)
Exposed
Low/Unexposed
6 months 20
12 months 3 7
2 years 2 11
4 years 4 5
Total 29 43

16. Aim 4: Telemedicine
See Dysmorphology Core

17. Phase II Analyses
Dr. Keen UC Davis has finished analysis of all 1,077 samples from the 10 shipments for Zn, Cu, Fe, Mg, Ca, transferrin receptor and ceruloplasmin activity
Samples through shipment 9 have been analyzed for homocysteine, C-reactive protein, ferritin and vitamin D
Choline and metabolites analyses have been completed for 802/1077 samples (74.5%)

18. Phase II Analyses: Dose (Number Binge Days) at Conception vs
Phase II Analyses: Dose (Number Binge Days) at Conception vs. Features FASD
Feature
Control
N = 256
0 days/wk
n =71
1 day/wk
n = 82
2 days/wk
n = 39
3+ days/wk
n = 24
Four-group
Alcohol Exposed
p-value
Five-group
PF <10th
20 (7.8%)
15 (21.1%)
17 (21.0%)
12 (30.8%)
6 (25.0%)
0.640
<0.001
Smooth philtrum
28 (10.9%)
10 (14.1%)
8 (9.8%)
6 (15.4%)
7 (29.2%)
0.139
0.104
Thin
vermilion
48 (18.8%)
18 (25.4%)
10 (12.2%)
11 (45.8%)
0.003
0.002
OFC <10th
14 (5.5%)
7 (9.9%)
12 (14.6%)
8 (20.5%)
0.116
FAS Dx on features
5 (2.0%)
7 (10.0%)
3 (3.7%)
5 (12.8%)
5 (20.8%)
0.055
6M MDI <85
28/205 (13.7%)
16/56
(28.6%)
18/64
(24.1%)
10/25
(40.0%)
13/24 (54.2%)
0.097
6M PDI <85
47/204 (23.0%)
18/56
(32.1%)
24/64
(37.5%)
9/24
0.893
0.079

19. Phase II Analyses: Dose (Drinks per Week) Mid-Pregnancy vs
Phase II Analyses: Dose (Drinks per Week) Mid-Pregnancy vs. Features FASD
Feature
Control
N = 256
<3 drinks/wk
n = 182
3-7 drinks/wk
n = 16
8+ drinks/wk
Three-group
Alcohol Exposed
p-value
Four-group
PF <10th
20 (7.8%)
32 (17.7%)
6 (37.5%)
12 (75.0%)
<0.001
Smooth philtrum
28 (10.9%)
21 (11.5%)
3 (18.8%)
7 (43.8%)
0.004
Thin
vermilion
48 (18.8%)
36 (19.8%)
5 (31.2%)
10 (62.5%)
OFC <10th
14 (5.5%)
24 (13.2%)
FAS Dx on features
5 (2.0%)
8 (4.4%)
9 (56.2%)
6M MDI <85
28/205
(13.7%)
38/142
(26.8%)
7/12
(58.3%)
10/13
(76.9%)
6M PDI <85
47/204
(23.0%)
46/142
(32.4%)
6/12
(50.0%)
9/13
(69.2%)
0.020

20. Alcohol Low/Unexposed Group
Phase II Analyses: Brain Ultrasound Measures as Predictors of 6 month Bayley Scores
Bayley
6 Mos
Ultrasound Measure N
Alcohol Exposed Group
Alcohol Low/Unexposed Group
Estimate SE
p-value
MDI
TCD
105
-0.07
0.65
0.916
172
0.11
0.34
0.746
OFD
104
0.43
0.23
0.069
171
-0.05
0.14
0.723
CCD
103
-0.47
0.45
0.300
0.40
0.080
FTD
0.51
0.21
0.018
0.47
0.13
<0.001
OOD
0.48
0.326
170
0.67
0.26
0.010
IOD
-1.22
0.46
174
-1.12 OD
1.21
0.85
0.159
-0.26
0.52
0.614
Each measure adjusted for gestational age at ultrasound, and each model for one or more of the following that met criteria as a confounder: maternal age, parity, socioeconomic status, education, pre—pregnancy BMI, vitamin use and smoking

21. Publication Progress
Chambers et al, Alcoholism Clinical Experimental Research, accepted 9/2013

22. for vitamin use in 1 mo before
Alcohol use 3 mo prior to pregnancy and multivitamin use 1 mo prior in 111,644 U.S. women in PRAMS
Alcohol in 3 mo before pregnancy OR
for vitamin use in 1 mo before
95% CI
p for trend
Number of drinks in average week
None
<=3
4-6
7-13
>=14
1.00
0.76
0.60
0.49
0.39
0.72, 0.80
0.56, 0.66
0.44, 0.55
0.32, 0.47
<0.001
Number of binge episodes (5 or more) 1
2-3
4-5
>=6
0.66
0.56
0.50
0.71, 0.81
0.62, 0.70
0.50, 0.64
0.44, 0.56
Weiss LA & Chambers CD, Alcohol Clin Exp Res, 2013;37: ; adjusted for education, race/ethnicity, income, age, parity

24. Publications Progress
Balaraman S, Lunde ER, Sawant O, Cudd TA, Washburn SE and Miranda RC. Maternal and Neonatal Plasma MicroRNA Biomarkers for Fetal Alcohol Exposure in an Ovine Model. Alcoholism: Clinical and Experimental Research, 2014 epub
Uriu-Adams JY, Obican SG, and Keen CL. Vitamin D and maternal and child health: Overview and implications for dietary requirements. Birth Defects Research. Part C: Embryo Today, 2013;99:24-44

25. Plans
Accelerate recruitment of children for the 2 and year old visits in the Spring and Summer months in Rivne and Khmelnytsky to meet recruitment objective of 90 children per group in July, 2014
Perform initial analysis on quality of biological samples from children
Generate preliminary results from Aim 3 and the genetic/epigenetic portion of Aim 4
Provide additional 3D images to Drs. Foroud and Hammond by moving the camera from site to site

26. Plans Continue in progress submissions for publication
Baseline minerals
Baseline vitamins
Baseline methyl donors
Intervention: 6 month and 12 month analyses
Pre/postnatal growth trajectory by maternal nutritional status
Ultrasound findings
Physical features in relation to dose
Retinol levels at baseline and inner canthal distance
Continue planned additional publications